The field of this invention is proteins which regulate cell function, and in particular, antagonize bone morphogenetic proteins.
Natural regulators of cellular growth, differentiation and function have provided important pharmaceuticals, clinical and laboratory tools, and targets for therapeutic intervention. A variety of such regulators have been shown to have profound effects on basic cellular differentiation and developmental pathways. For example, the recently cloned cerberus protein induces the formation of head structures in anterior endoderm of vertebrate embryos. Similarly, the Noggin protein induces head structures in vertebrate embryos, and can redirect mesodermal fates from ventral fates, such as blood and mesenchyme, to dorsal fates such as muscle and notochord and can redirect epidermal fates to anterior neural fates. The activities of chordin are similar to those of Noggin, reflecting a common mechanism of actionxe2x80x94namely antagonizing bone morphogenetic proteins (BMP) and thereby preventing their function. BMPs have diverse biological activities in different biological contexts, including the induction of cartilage, bone and connective tissue, and roles in kidney, tooth, gut, skin and hair development.
Different members of the TGFxcex2 superfamily can instruct cells to follow different fates, for example TGFxcex2 induces neural crest to form smooth muscle, while BMP2 induces the same cells to become neurons. In Xenopus experiments, dissociated animal cap cells (prospective ectoderm) become epidermis in response to BMP4 but become mesoderm in response to activin.
Since the sequence identity between activin and BMP4 is low, it is not surprising that they induce different fates. It is more surprising that members of the BMP subfamily, which are quite closely related in sequence, can induce distinct fates. A striking example results from implantation of a matrix impregnated with a BMP into muscle; when the effects are monitored histologically, BMP2, 4 and 7 induce endochondral bone formation, whereas a related molecule BMP12/GDF7 induces connective tissue similar to tendon. Similarly, BMP4 can induce cell death in the hindbrain neural crest, while the related protein dorsalin does not.
Since different BMP family members can induce different fates, then BMP antagonists that have specificity in blocking subsets of BMPs could change the balance of BMPs that are presented to a cell, thus altering cell fate. In view of the importance of relative BMP expression in human health and disease, regulators of cellular function and BMP function in particular, such as Noggin and cerberus, provide valuable reagents with a host of clinical and biotechnological applications. The present invention relates to a new family of regulators of cellular function.
Bouwmeester, et al. (1996) Nature 382: 595-601 describe the cloning of Xenopus cerberus gene; Lamb, T. M., et al. (1993) Science 262: 713-718; Smith, W. C., et al. (1992) Cell 70: 829-840; Smith, W. C., et al. (1993) Nature 361: 547-549; and Zimmerman, L. B., et al. (1996) Cell 86: 599-606 describe the isolation and function of the Noggin protein. Piccolo, S., et al. (1996) Cell 86: 589-598; Sasai, Y., et al. (1995) Nature 376: 333-336; and Sasai, Y., et al. (1994) Cell 79: 779-790 relate to the chordin protein. Enomoto et al. (1994) Oncogene 9: 2785-2791 and Ozaki, et al. (1996) Jpn. J. Cancer Res. 87: 58-61 describe human and murine homologs of the DAN gene. Hsu, et al. (1998) Mol Cell 1:673-683 describing Gremlin from a variety of species, including human; Minabe-Saegusa, C., et al. (1998) Dev Growth Differ 40:343-353 which describes mouse PRDC.
The invention provides methods and compositions relating to DCR5, a protein related to Gremlin, DAN (Differential-screening-selected gene Aberrative in Neuroblastoma) and Cerberus, and related nucleic acids. Included are natural DCR5 homologs from different species, as well as proteins comprising a DCR5 domain and having DCR5-specific activity, particularly the ability to antagonize a bone morphogenetic protein. The proteins may be produced recombinantly from transformed host cells with the subject nucleic acids. The invention provides isolated hybridization probes and primers capable of specifically hybridizing with the disclosed genes, specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g., genetic hybridization screens for DCR5 transcripts), therapy (e.g., gene therapy to modulate DCR5 gene expression) and in the biopharmaceutical industry (e.g., reagents for screening chemical libraries for lead pharmacological agents).
Preferred applications of the subject DCR5 proteins include modifying the physiology of a cell comprising an extracellular surface by contacting the cell or medium surrounding the cell with an exogenous DCR5 protein under conditions whereby the added protein specifically interacts with a component of the medium and/or the extracellular surface to effect a change in the physiology of the cell. Also preferred are methods for screening for biologically active agents, which methods involve incubating a DCR5 protein in the presence of an extracellular DCR5 protein-specific binding target and a candidate agent, under conditions whereby, but for the presence of the agent, the protein specifically binds the binding target at a reference affinity; detecting the binding affinity of the protein to the binding target to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that the agent modulates the binding of the protein to the binding target.
Another preferred embodiment of the invention is a method of treatment of a human or animal body by administering a therapeutic dosage of a human DCR5 polypeptide as wherein the treatment is regulation of cartilage and bone growth.
An additional preferred embodiment of the invention is a ligandbody which comprises human DCR5 fused to an immunoglobulin constant region, wherein the immunoglobulin constant region is the Fc portion of human IgG1.
In a preferred embodiment, a ligandbody may be used in a method of treatment of the human or animal body, or in a method of diagnosis.
The invention provides DCR5 proteins which include natural DCR5 proteins and recombinant proteins comprising a DCR5 amino acid sequence, or a functional DCR5 protein domain thereof having an assay-discemable DCR5-specific activity. Accordingly, the proteins may be deletion mutants of the disclosed natural DCR5 proteins and may be provided as fusion products, e.g., with non-DCR5 polypeptides. The subject DCR5 protein domains have DCR5-specific activity or function and are functionally distinct from each other and from DAN, cerberus, Gremlin and Noggin homologs. Such domains include at least 6 and preferably at least 8 consecutive residues of a natural DCR5 protein (See DAN sequence reported by Enomoto, et al. (1994) Oncogene 9: 2785-2791). Preferred DCR5 proteins comprise a DCR5 sequence conserved across species.
The DCR5 proteins described herein are structurally and functionally related to DAN and Cerberus in that they are extracellularly active as antagonists of certain morphogenetic proteins such as BMPs. DCR5-specific activity or function may be determined by convenient in vitro cell-based, or in vivo assaysxe2x80x94e.g., in vitro binding assays, cell culture assays, in animals (e.g., immune response, gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the specific molecular interaction of a DCR5 protein with a binding target is evaluated. The binding target may be a natural binding target such as a TGFxcex2 protein, a morphogenetic protein, preferably a bone morphogenetic protein such as BMP2 or BMP4, a chaperon, or other regulator that directly modulates DCR5 activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or a DCR5 specific agent such as those identified in assays described below. Generally, binding specificity is assayed by bioassay (e.g., the ability to induce neuronal tissue from injected embryonic ectoderm), TGFxcex2 protein binding equilibrium constants (usually at least about 107 Mxe2x88x921, preferably at least about 108 Mxe2x88x921, more preferably at least about 109 Mxe2x88x921), by the ability of the subject protein to function as negative mutants in DCR5-expressing cells, to elicit DCR5 specific antibody in a heterologous host (e.g., a rodent or rabbit), etc.
The claimed proteins may be isolated or purexe2x80x94an xe2x80x9cisolatedxe2x80x9d protein is one that is no longer accompanied by some of the material with which it is associated in its natural state, and that preferably constitutes at least about 0.5%, and more preferably at least about 5% by weight of the total protein in a given sample; a xe2x80x9cpurexe2x80x9d protein constitutes at least about 90%, and preferably at least about 99% by weight of the total protein in a given sample. The subject proteins and protein domains may be synthesized, produced by recombinant technology, or purified from cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g., Molecular Cloning, A Laboratory Manual (Sambrook, et al., Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY).
The subject proteins find a wide variety of uses including use as immunogens, targets in screening assays, bioactive reagents for modulating cell growth, differentiation and/or function, etc. For example, the invention provides methods for modifying the physiology of a cell comprising an extracellular surface by contacting the cell or medium surrounding the cell with an exogenous DCR5 protein under conditions whereby the added protein specifically interacts with a component of the medium and/or the extracellular surface to effect a change in the physiology of the cell. According to these methods, the extracellular surface includes plasma membrane-associated receptors; the exogenous DCR5 refers to a protein not made by the cell or, if so, expressed at non-natural levels, times or physiologic locales; and suitable media include in vitro culture media and physiological fluids such as blood, synovial fluid, etc. Effective administrations of subject proteins can be used to reduce undesirable (e.g., ectopic) bone formation, inhibit the growth of cells that require a morphogenetic protein (e.g., BMP-dependent neuroblastomas and gliomas), alter morphogen-dependent cell fate/differentiation in culture, such as with cells for transplantation or infusion, etc. The proteins may be introduced, expressed, or repressed in specific populations of cells by any convenient way such as microinjection, promoter-specific expression of recombinant enzyme, targeted delivery of lipid vesicles, etc.
The invention provides natural and non-natural DCR5-specific binding agents, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. DCR5-specific binding agents include DCR5-specific ligands such as BMPs, and receptors, such as somatically recombined protein receptors like specific antibodies or T-cell antigen receptors (See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and also includes other natural binding agents identified with assays such as one-, two- and three-hybrid screens, and non-natural binding agents identified in screens of chemical libraries such as described below. Agents of particular interest modulate DCR5 function.
The invention provides DCR5 nucleic acids, which find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc., as well as use in detecting the presence of DCR5 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional DCR5 homologs and structural analogs.
The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e., no longer accompanied by some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Nucleic acids comprising the nucleotide sequence of SEQ ID NO. 11 or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by a sequence other than that to which it is joined on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is immediately flanked by a sequence other than that to which it is joined on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.
The amino acid sequences of the disclosed DCR5 proteins are used to back translate DCR5 protein-encoding nucleic acids optimized for selected expression systems (Holler, et al. (1993) Gene 136: 323-328; Martin, et al. (1995) Gene 154: 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural DCR5 encoding nucleic acid sequences (xe2x80x9cGCGxe2x80x9d software, Genetics Computer Group, Inc., Madison, Wis.). DCR5 encoding nucleic acids may be part of expression vectors and may be incorporated into recombinant host cells, e.g., for expression and screening, for transgenic animals, for functional studies such as the efficacy of candidate drugs for disease associated with DCR5 mediated signal transduction, etc. Expression systems are selected and/or tailored to effect DCR5 protein structural and functional variants through alternative post-translational processing.
The invention also provides for nucleic acid hybridization probes and replication/amplification primers having a DCR5 cDNA specific sequence and sufficient to effect specific hybridization with SEQ ID NO. 11. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5xc3x97SSPE (0.18 M NaCl, 0.01 M NaPO4, pH7.7, 0.001 M EDTA) buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5xc3x97SSPE buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE buffer at 42xc2x0 C. DCR5 cDNA homologs can also be distinguished from other protein using alignment algorithms, such as BLASTX (Altschul, et al. (1990) Basic Local Alignment Search Tool, J. Mol. Biol. 215: 403-410).
DCR5 hybridization probes find use in identifying wild-type and mutant alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. DCR5 nucleic acids are also used to modulate cellular expression or intracellular concentration or availability of active DCR5. DCR5 inhibitory nucleic acids are typically antisensexe2x80x94single stranded sequences comprising complements of the disclosed natural DCR5 coding sequences. Antisense modulation of the expression of a given DCR5 protein may employ antisense nucleic acids operably linked to gene regulatory sequences. Cells are transfected with a vector comprising a DCR5 sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to endogenous DCR5 encoding mRNA. Transcription of the antisense nucleic acid may be constitutive or inducible and the vector may provide for stable extrachromosomal maintenance or integration. Alternatively, single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding a given DCR5 protein may be administered to the target cell, in or temporarily isolated from a host, at a concentration that results in a substantial reduction in expression of the targeted protein. An enhancement in DCR5 expression is effected by introducing into the targeted cell type DCR5 nucleic acids which increase the functional expression of the corresponding gene products. Such nucleic acids may be DCR5 expression vectors, vectors which upregulate the functional expression of an endogenous allele, or replacement vectors for targeted correction of mutant alleles. Techniques for introducing the nucleic acids into viable cells are known in the art and include retroviral-based transfection, viral coat protein-liposome mediated transfection, etc.
The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of DCR5 modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate DCR5 interaction with a natural DCR5 binding target. A wide variety of assays for binding agents are provided including protein-protein binding assays, immunoassays, cell based assays, etc. Preferred methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds.
In vitro binding assays employ a mixture of components including a DCR5 protein, which may be part of a fusion product with another peptide or polypeptide, e.g., a tag for detection or anchoring, etc. The assay mixtures comprise a natural DCR5 binding target, e.g., a TGFxcex2 protein such as a BMP. While native binding targets may be used, it is frequently preferred to use portions thereof as long as the portion provides binding affinity and avidity to the subject DCR5 conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds, preferably small organic compounds, and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents such as salts, buffers, neutral proteins, e.g., albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may also be included. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. The mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the DCR5 specifically binds the cellular binding target, portion or analog with a reference binding affinity. Incubation periods are chosen for optimal binding but are also minimized to facilitate rapid, high throughput screening.
After incubation, the agent-biased binding between the DCR5 and one or more binding targets is detected by any convenient way. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. Separation may be effected by precipitation, immobilization, etc., followed by washing by, e.g., membrane filtration or gel chromatography. For cell-free binding assays, one of the components usually comprises or is coupled to a label. The label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc., or indirect detection such as an epitope tag, an enzyme, etc. A variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g., through optical or electron density, radiative emissions, nonradiative energy transfers, or indirectly detected with antibody conjugates, etc. A difference in the binding affinity of the DCR5 protein to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the DCR5 protein to the corresponding binding target. A difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.
The invention provides for a method for modifying the physiology of a cell comprising an extracellular surface in contact with a medium, said method comprising the step of contacting said medium with an exogenous DCR5 protein under conditions whereby said protein specifically interacts with at least one of a component of said medium and said extracellular surface to effect a change in the physiology of said cell.
The invention further provides for a method for screening for biologically active agents, said method comprising the steps of a) incubating a DCR5 protein in the presence of an extracellular DCR5 protein specific binding target and a candidate agent, under conditions whereby, but for the presence of said agent, said protein specifically binds said binding target at a reference affinity; b) detecting the binding affinity of said protein to said binding target to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that said agent modulates the binding of said protein to said binding target.
The invention also provides for the production of ligandbodies. Ligandbodies are comprised of a ligand polypeptide coupled to the Fc domain of IgG and are able to dimerize (see for example Davis, et al., 1994, Science 266:816-819). Ligandbodies have the advantage of exhibiting enhanced pharmacokinetic properties. Thus, DCR5 ligandbodies may be useful in therapeutic applications where enhanced pharmacokinetic properties of DCR5 is desirable.
One embodiment of the invention is an isolated DCR5 protein comprising the amino acid sequence as set forth in SEQ ID NO. 12 or a fragment thereof having DCR5-specific activity.
Another embodiment of the invention is a recombinant nucleic acid encoding DCR5 protein comprising the amino acid sequence as set forth in SEQ ID NO. 12 or a fragment thereof having DCR5-specific activity.
Still another embodiment is an isolated nudeic acid comprising a nucleotide sequence as set forth in SEQ ID NO. 11 or a fragment thereof having at least 18 consecutive bases of SEQ ID NO. 11 and sufficient to specifically hybridize with a nucleic acid having the sequence of SEQ ID NO. 11 in the presence of natural DAN and cerberus cDNA.
Another preferred embodiment of the invention is a method of treatment of a human or animal body by administering a therapeutic dosage of a human DCR5 polypeptide as wherein the treatment is regulation of cartilage and bone growth.
The present invention also provides for antibodies to the DCR5 protein described herein which are useful for detection of the protein in, for example, diagnostic applications. For preparation of monoclonal antibodies directed toward this DCR5 protein, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in xe2x80x9cMonoclonal Antibodies and Cancer Therapy,xe2x80x9d Alan R. Liss, Inc. pp. 77-96) and the like are within the scope of the present invention.
The monoclonal antibodies for diagnostic or therapeutic use may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982, Meth. Enzymol. 92:3-16). Chimeric antibody molecules may be prepared containing a mouse antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).
Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of the DCR5 protein described herein. For the production of antibody, various host animals can be immunized by injection with the DCR5 protein, or a fragment or derivative thereof, including but not limited to rabbits, mice and rats. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund""s (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, polypeptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. 
A molecular clone of an antibody to a selected DCR5 protein epitope can be prepared by known techniques. Recombinant DNA methodology (see e.g., Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof.
The present invention provides for antibody molecules as well as fragments of such antibody molecules. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(abxe2x80x2)2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fabxe2x80x2 fragments which can be generated by reducing the disulfide bridges of the F(abxe2x80x2)2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. Antibody molecules may be purified by known techniques, e.g., immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof.
The invention further provides for a method of using a DCR5 protein or fragment thereof as an antagonist of the activity of a bone morphogenetic protein (BMP), either alone, or in combination with other factors, including DAN, Cerberus, b57 or von Willebrand factor to regulate or modulate the activity of a BMP.